The 4 kDa amyloid ?-protein (A?) is strongly implicated in the pathogenesis of Alzheimer's disease (AD). A? is produced through successive proteolysis of the type I integral membrane amyloid ?-protein precursor (APP), first on the lumen/extracellular side by the membrane-tethered ?-secretase and then within the transmembrane domain (TMD) by ?-secretase, a membrane-embedded aspartyl protease complex that contains the multi-pass presenilin as the catalytic component. Cleavage by ?-secretase determines the length of the A? peptide at the C-terminus, with longer forms of A? containing more of the TMD and being much more prone to self-assembly into neurotoxic aggregates. Although ?-secretase is considered an important target for the development of AD therapeutics, the enzyme cleaves a variety of other type I integral membrane proteins, most notably Notch receptors. Proteolysis of Notch with attendant release of its intracellular domain is part of a signaling pathway that is central to many types of cell differentiation events, and inhibition of this pathway with ?-secretase inhibitors (GSIs) results in severe toxicities that are unacceptable for an AD drug. Nevertheless, the discovery of GSIs that are selective for APP over Notch (so-called Notch-sparing GSIs) has led to the advancement of several candidates into clinical trials. However, the means by which these compounds selectively inhibit the protease complex are unknown, and an understanding of their mechanisms of selectivity might reveal strategies for developing more selective agents. The goal of this project is to identify substrate and enzyme determinants that confer selectivity for APP vis-?-vis Notch of Notch-sparing ?-secretase inhibitors. With this goal in mind, specific aims are proposed to address the following questions: (1) What regions/residues of APP and Notch confer substrate selectivity of Notch-sparing GSIs? (2) What is the potency and selectivity of Notch-sparing GSIs for ?-secretase carrying familial AD mutations? (3) What is the potency and selectivity of Notch-sparing GSIs for PS1 vs. PS2 ?-secretase? The identification of substrate and enzyme determinants of APP/Notch selectivity for these compounds should provide important information relevant to where these compounds bind and how that binding might result in allosteric changes that confer substrate selectivity.